The invention relates generally to molecular separation techniques, and particularly to techniques for identifying oligonucleotides separated by gel electrophoresis.
Many procedures in molecular biology require that heterogeneous mixtures of DNA or RNA be electrophoretically separated into homogeneous components according to mass, charge, conformation, isoelectric point, or the like. The homogeneous components are then detected by densitometry or by radioactive, fluorescent, or chromogenic labeling. Each such method of identification has its own advantages and disadvantages, e.g. Gould and Matthews, Separation Methods for Nucleic Acids and Oligonucleotides (North-Holland Publishing Company, Amsterdam, 1976) pgs. 337-344. For example, until recently DNA sequencing techniques relied exclusively on radioactive labels for distinguishing oligonucleotides separated by electrophoresis. Radioactive labels are highly sensitive, and can be readily incorporated into the molecules of interest. However, there are several inherent disadvantages to their use: In autoradiography resolution is limited by the omnidirectional nature of the tracks of the decay particles, the thickness and distance of the autoradiographic emulsion, and the cumulative nature of the signal recorded in the emulsion. Radioactive labels pose a laboratory health hazard, which requires that the labels receive special handling and disposal. And, finally, radioactive labels require long exposure or counting times for adequate signal to noise resolution. This latter disadvantage is especially acute when labels are used in conjunction with automated techniques, such as automated DNA sequencing where bands of different kinds of labeled nucleotides must be rapidly identified as they traverse a single electrophoresis lane. Not only are there no nucleotide-specific radioactive labels for practical identification, but even if there were, current detection techniques such as autoradiography or scintillation counting are too time consuming. As a consequence, fluorescent labeling means have been sought for use with DNA sequencing techniques.
Fluorescent labels can be detected immediately after application; they are conveniently handled; and they permit the precise localization and quantification of the labeled molecules.
Several factors constrain the selection of fluorescent labels for an oligomeric series undergoing separation by gel electrophoresis, such as an oligomeric series of nucleotides whose members differ only in base number. First, the labels must not adversely affect electrophoretic mobility so that extensive band broadening occurs. Nor can the relative effects of the labels on electrophoretic mobility be such that one or more band positions become reversed or overlapping thereby destroying the correspondence between band ordering and the natural order of the oligomeric series. Unfortunately there is no reliable way to predict with certainty the electrophoretic behavior of an oligomer with an arbitrarily chosen label attached, such as an organic dye. Procedures for electrophoretic separations are usually arrived at empirically; however, two major factors determining electrophoretic mobility are charge and molecular weight. Other important factors include configuration of the oligomers and gel polymer density, Gould and Matthews, Separation Methods for Nucleic Acids and Oligonucleotides (North-Holland Publishing Company, Amsterdam, 1976), p. 313.
Second, where several distinct labels are required, a selection of dyes cannot have significantly overlapping emission bands. However, given that emission band halfwidth for organic fluorescent dyes is typically about 40-80 nanometers and that the width of the visible spectrum is only about 350-400 nanometers, it is exceedingly difficult to find a suitable selection of fluorescent dyes without significant overlap whenever three or more distinct fluorescent labels are required. Moreover, when several fluorescent dyes are used, excitation becomes difficult because the absorption bands of the dyes are often widely separated. The most efficient excitation occurs when each dye is illuminated at the wavelength corresponding to its absorption band maximum. When several dyes are used together one is often forced to make a trade off between the sensitivity of the detection system and the increased cost of providing separate excitation sources for each dye. Finally, the fluorescent labels must be compatible with the chemistry used to create or manipulate the molecules which are labeled. For example, in enzymatic sequencing of DNA, the fluorescent dyes used to label primers cannot interfere with DNA polymerase activity.
Smith et al, in "Synthesis of Oligonucleotides Containing an Aliphatic Amino Group at the 5' Terminus: Synthesis of Fluorescent DNA Primers for Use in DNA Sequence Analysis," Nucleic Acids Research, Vol. 13, pgs. 2399-2412 (1985), disclose a set of four fluorescent dyes for use in enzymatic DNA sequence analysis for labeling oligonucleotides separated by electrophoresis. Each dye from the set is used to identify on an electrophoresis gel bands of oligonucleotides having the same 3' terminal nucleotide.